The present invention relates to a microfluidic device, which can be interfaced to a mass spectrometer (MS). The device comprises a microchannel structure, which has a first port (inlet port) and a second port (outlet port). A sample to be analysed is applied to the first port and presented to the mass spectrometer in the second port. This second port will be called an MS-port. There may be additional inlet and outlet ports, and also additional identical or similar microchannnel structures. During passage through the microchannel structure the sample is prepared to make it suitable for analysis by mass spectrometry.
The sample presented in an MS-port will be called an MS-sample. An analyte in an MS-sample is an MS-analyte. xe2x80x9cSamplexe2x80x9d and xe2x80x9canalytexe2x80x9d without prefix will primarily refer to a sample applied to an inlet port.
Conductive and non-conductive properties are with respect to conducting electricity.
The present invention concerns mass spectrometry in which the MS-samples are subjected to Energy Desorption/Ionisation from a surface by input of energy (EDI MS). Generically this kind of process will be called EDI and the surface an EDI-surface in the context of the invention. Typically EDIs are thermal desorption/ionisation (TDI), plasma desorption/ionisation (PDI) and various kinds of irradiation desorption/ionisation (IDI) such as by fast atom bombardment (FAB), electron impact etc. In the case a laser is used the principle is called laser desorption/ionisation (LDI). Desorption may be assisted by presenting the MS analyte together with various helper substances or functional groups on the surface. Common names are matrix assisted laser desorption/ionisation (MALDI) including surface-enhanced laser desorption/ionisation (SELDI). For MALDI see the publications discussed under Background Publications below. For SELDI see WO 0067293 (Ciphergen Biosystems).
The term xe2x80x9cEDI-areaxe2x80x9d comprises the EDI-surface as such and the part of a substrate covered by this surface, e.g. the part of the substrate that is under the EDI-surface. Compare the description of FIG. 4.
The term xe2x80x9cmicroformatxe2x80x9d means that in at least a part of a microchannel structure the depth and/or width is in the microformat range, i.e.  less than 103 xcexcm, preferably  less than 102 xcexcm. The depth and/or width are within these ranges essentially everywhere between an inlet port and an outlet port, e.g. between a sample inlet port and an MS-port. The term xe2x80x9cmicrochannel structuresxe2x80x9d includes that the channels are enclosed in a substrate.
The term xe2x80x9cmicrofluidic devicexe2x80x9d means that transport of liquids and various reagents including analytes are transported between different parts within the microchannel structures by a liquid flow.
For some time there has been a demand for microfluidic sample handling and preparation devices with integrated MS-ports. This kind of devices would facilitate automation and parallel experiments, reduce loss of analyte, increase reproducility and speed etc.
WO 9704297 (Karger et al) describes a microfluidic device that has an outlet port that is claimed useful when conducting electrospray ionisation mass spectrometry (ESI MS), atmospheric pressure chemical ionisation mass spectrometry (APCI MS), matrix assisted laser desorption/ionisation mass spectrometry (MALDI MS) and a number of other analytical principles.
U.S. Pat. No. 5,705,813 (Apffel et al) and U.S. Pat. No. 5,716,825 (Hancock et al) describe a microfluidic chip containing an MS-port. After processing a sample within the chip the sample will appear in the MS-port. The whole chip is then placed in an MALDI-TOF MS apparatus. The microfluidic device comprises
(a) an open ionisation surface that may be used as the probe surface in the vaccum gate of an MALDI-TOF MS apparatus (column 6, lines 53-58 of U.S. Pat. No. 5,705,813), or
(b) a pure capture/reaction surface from which the MS-analyte can be transferred to a proper probe surface for MALDI-TOF MS (column 12, lines 13-34, of U.S. Pat. No. 5,716,825).
These publications suggest that means for transporting the liquid within a microchannel structure of the device are integrated with or connected to the device. These means are electrical connections, pumps etc, which impose an extra complexity on the design and use and may negatively influence the production costs, easiness of handling etc.
Although both U.S. Pat. No. 5,705,813 (Apffel et al) and U.S. Pat. No. 5,716,825 (Hancock et al) explicitly concern microfluidic devices, they are scarce about
the proper fluidics around the MALDI ionisation surface,
the proper crystallisation on the MALDI ionisation surface,
the proper geometry of the port in relation to crystallisation, evaporation, the incident laser beam etc,
the conductive connections to the MALDI ionisation surface for MALDI MS analysis.
These features are important in order to manage with interfacing a microfluidic device to an MALDI mass spectrometer.
WO 9704297 (Karger et al) and WO 0247913 (Gyros A B) suggest a radial or spoke arrangement of the microchannel structures of a microfluidic device.
WO 9721090 (Mian et al) (page 30, lines 3-4, and page 51, line 10) and WO 0050172 (Burd Mehta) (page 55, line 14) suggest in general terms that their microfluidic systems might be used for preparing samples that are to be analysed by mass spectrometry. WO 9721090 is explicitly related to a system in which centrifugal force is used for driving the liquid flow.
A number of publications referring to the use of centrifugal force for moving liquids within microfluidic systems have appeared during the last years. See for instance WO 9721090 (Gamera Bioscience), WO 9807019 (Gamera Bioscience) WO 9853311 (Gamera Bioscience), WO 9955827 (Gyros A B), WO 9958245 (Gyros A B), WO 0025921 (Gyros A B), WO 0040750 (Gyros A B), WO 0056808 (Gyros A B), WO 0062042 (Gyros A B), WO 0102737 (Gyros A B), WO 0147637, (Gyros A B), WO 0154810 (Gyros A B), WO 0147638 (Gyros A B), WO 0146465 (Gyros A B).
U.S. Ser. No. 60/315,471 and the corresponding International Patent Application WO 02074438 discuss various designs of microfluidic functions, some of which can be applied to the present invention.
Kido et al., (xe2x80x9cDisc-based immunoassay microarraysxe2x80x9d, Anal. Chim. Acta 411 (2000) 1-11) has described microspot immunoassays on a compact disc (CD). The authors suggest that a CD could be used as a continuous sample collector for microbore HPLC and subsequent detection for instance by MALDI MS. In a preliminary experiment a piece of a CD manufactured in polycarbonate was covered with gold and spotted with a mixture of peptides and MALDI matrix.
A first object is to provide improved means and methods for transporting samples, analytes including fragments and derivatives, reagents etc in microfluidic devices that are capable of being interfaced with a mass spectrometer that require energy desorption/ionisation of an MS-analyte from a surface by input of energy.
A second object is to provide improved microfluidic methods and means for sample handling before presentation of a sample analyte as an MS-analyte. Sub-objects are to provide an efficient concentration, purification and/or transformation of a sample within the microfluidic device while maintaining a reproducible yield/recovery, and/or minimal loss of precious material.
A third object is to provide improved microfluidic methods and means that will enable efficient and improved presentation of an MS-sample/MS-analyte. This object applies to MS-samples that are presented on an EDI-surface.
A fourth object is to enable reproducible mass values from an MS-sample that is presented on an EDI surface that is present in a microfluidic device
A fifth object is to provide improved microfluidic means and methods for parallel sample treatment before presentation of the MS-analyte from an EDI-surface to mass spectrometry. The improvements of this object refer to features such as accuracy in concentrating, in chemical transformation, in required time for individual steps and for the total treatment protocol etc. By parallel sample treatment is meant that two or more sample treatments are run in parallel in different microchannel structures within the same microfluidic device. The number of parallel runs may be more than five, such as more than 10, 50, 80, 100, 200, 300 or 400 runs. Particular important numbers of parallel samples are below or equal to the standard number of wells in microtiter plates, e.g. 96 or less, 384 or less, 1536 or less, etc
A sixth object is to provide a cheap and disposable microfluidic device unit enabling parallel sample treatments and having one or more MS-ports that are adapted to a mass spectrometer that require energy desorption/ionisation of an MS-analyte from a surface by input of energy.
The present inventors have recognized that the optimisation of an EDI-area in a microfluidic device is related to
(a) the design and/or positioning of a conductive layer in the EDI-area, and/or
(b) the need of a calibrator area associated with an EDI MS-port, and/or
(c) the need of a proper conductive connection to the EDI-area for MS analysis.
The conductive connection will support the proper voltage and/or charge transport at the EDI-area, for instance. Improper conductive properties may negatively affect the mass accuracy, sensitivity, resolution etc. The importance of (a)-(c) increases if there is a plurality of microchannel structures in the microfluidic device.
The present inventors have also recognized that several of the above-mentioned objects can be met in the case inertia force is used for transportation of a liquid within a microfluidic device as defined in this specification. This is applicable to liquid, such as washing liquids and liquids containing at least one of (a) the analyte including derivatives and fragments thereof, (b) a reagent used in the transformation of the sample/analyte, etc.
The first aspect of the invention is a microfluidic device in form of a disc comprising an MS-port for presentation of an MS-analyte to an EDI-MS apparatus. The MS-port is a part of a microchannel structure (I) which comprises an inlet port for a sample. The MS-port also comprises an EDI-area with a conductive layer (I) and an EDI-surface from which the MS-analyte is to be desorbed/ionised. The disc is characterized in that layer (I) has a conductive connection and/or that there is a calibrator area in the proximity of said MS-port.
The MS-port typically is in the form of a wall or depression with an opening to ambient atmosphere and in fluid communication microchannel structure (I). As discussed in more detail below the disc may comprise two or more of microchannel structure (I), i.e. a plurality of them. Layer (I) may be placed at different positions in the EDI-area.
A second aspect of the invention is a method for transforming a liquid sample containing an analyte to an MS-sample containing an MS-analyte and presenting the MS-sample to a mass spectrometer. The method is characterized in comprising the steps of:
(a) providing a microfluidic device as defined in this specification,
(b) applying the liquid sample to an inlet port of one or more of the covered microchannel structures of the microfluidic device,
(c) transforming the liquid sample to an MS-sample containing the MS-analyte within at least one the microchannel structures to which a sample has been applied in step (b), and
(d) presenting the MS-analyte to the mass spectrometer.
A variant of the second aspect is a method for collecting mass spectrometric data of an analyte or an analyte-derived entity, for instance in order to gain molecular weight and structure information about an analyte. The analyte-derived entity is then formed in the innovative microfluidic device according to steps (a)-(d) in the preceding paragraph.
The various innovative embodiments of the invention are further defined in the text below including the claims.
Liquid Transport
The liquid flow used for transport of reagents, analyte, analyte derived entities etc within the microchannel structures may be driven by electrokinetic forces and/or by non-electrokinetic forces. Typical non-electrokinetic forces are inertia force, such as centrifugal force, capillary forces, forces created by pressure differences etc. The term xe2x80x9cforces created by pressure differencesxe2x80x9d includes hydrostatic pressure created within certain kinds of microchannel structures by the combined action of spinning and application of a series of liquid aliquots (see below and WO 0146465 (Gyros A B)).
In preferred variants, the liquid flow within the individual microchannel structures of a device is created by the application of inertia force. Inertia force may be the driving force in only a part of a microchannel structure or the whole way from an inlet port to an MS-port and/or to any other outlet port. It is believed that the most general and significant advantages of using inertia force will be accomplished in so called transporting zones, i.e. between zones having predetermined functionalities, or for overcoming or passing through valve functions within a microchannel structure (capillary junctions, hydrophobic breaks etc). See below.
At the priority date the most important inertia force to be used in the innovative devices is centrifugal force, i.e. spinning of the device in order to accomplish an outward radial transportation of liquid which is present in a microchannel structure that comprise parts at different radial distances from the spinning axis (axis of symmetry). The spinning axis is perpendicular to the plane of the disc. The disc/device is preferably circular and centrifugal force is used in at least a part of each microchannel structures, for instance to take the sample into an MS-port.
Inertia force, such as centrifugal force, may be combined with one or more other kinds of driving forces. The combination may be in the same part of a microchannel structure. The combination may also mean that inertia force is utilized for transport in a part where the flow shall be directed outwards towards the periphery of a circular disc and other forces in some other part for creating a flow inwards or more or less parallel to the periphery of a disc. Capillary force may typically be used to transport a liquid aliquot from an inlet port into a microchannel associated with the inlet port. This kind of microchannels may be directed inwards towards the centre of a disc or more or less perpendicular thereto.
It may be beneficial to include a pulse giving increased flow for over-coming inter-channel variations in flow resistance, in particular when initiating flow and/or when the liquid is to pass through branchings and curvatures.
The Sample.
The sample applied to an inlet port may contain one or more analytes, which may comprise lipid, carbohydrate, nucleic acid and/or peptide structure or any other organic structure. The analyte may also comprise an inorganic structure. The sample treatment protocol to take place within the microchannel structure typically means that the sample is transformed to one or more MS-samples in which
(a) the MS-analyte is a derivative of the starting analyte and/or
(b) the amount(s) of non-analyte species have been changed compared to the starting sample, and/or
(c) the relative occurrence of different MS-analytes in a sample is changed compared to the starting sample, and/or
(d) the concentration of an MS-analyte is changed relative the corresponding starting analyte in the starting sample, and/or
(e) sample constituents, such as solvents, have been changed and/or the analyte has been changed from a dissolved form to a solid form, for instance in a co-crystallised form.
Item (a) includes digestion into fragments of various sizes and/or chemical derivatization of an analyte. Digestion may be purely chemical or enzymatic. Derivatization includes so-called mass tagging of either the starting analyte or of a fragment or other derivative formed during a sample treatment protocol, which takes place in the microchannel structure. Items (b) and/or (c) include that the sample analyte has been purified and/or concentrated. Items (a)-(d), in particular, apply to analytes that are biopolymers comprising carbohydrate, nucleic acid and/or peptide structure.
The sample is typically in liquid form and may be aqueous.
The sample may also pass through a microchannel structure without being changed. In this case the processing within a microchannel structure only provides a form for dosing of the analyte to the mass spectrometer.